Detection system for high sensitivity fluorescent assays

ABSTRACT

This invention relates to a detection system for measuring a fluorescent signal in a fluorescent assay. The system comprises a probe having a small sensing surface bound with a fluorescent label, and a light source and a detector both mounted at the proximal side of the sensing surface of the substrate. The invention also relates to a method for detecting an analyte in a liquid sample using a probe tip having a small surface area (≤5 mm) and a high molecular weight polymer (≥1 MD) having multiple binding molecules and multiple fluorescent labels. The binding reaction is accelerated by flowing the reaction solutions laterally and moving the probe tip up and down in the reaction vessels. The invention furthers relates to a fluorescent labeling composition comprising a cross-linked FICOLL® molecule having a plurality of binding molecules and a plurality of fluorescent labels.

This application is a continuation of U.S. patent application Ser. No.13/846,517, filed Mar. 18, 2013; which is a continuation of U.S.application Ser. No. 13/653,362, filed Oct. 16, 2012, now U.S. Pat. No.8,415,172; which is a divisional of U.S. application Ser. No.13/224,174, filed Sep. 1, 2011, now U.S. Pat. No. 8,309,369; which is acontinuation of PCT/US2010/025938, filed Mar. 2, 2010; which claims thepriority of U.S. Provisional Application No. 61/209,116, filed Mar. 3,2009; 61/299,525, filed Jan. 29, 2010, and 61/303,567, filed Feb. 11,2010. The contents of the above-identified applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a detection system for measuring a fluorescentsignal in a fluorescent assay. The system comprises a probe having asensing surface bound with a fluorescent label, and a light source and adetector both mounted at the proximal side of the sensing surface of thesubstrate. The invention also relates to a method for detecting ananalyte in a liquid sample using a probe having a small surface and apolymer having multiple binding molecules and multiple fluorescentlabels. The invention furthers relates to a fluorescent labelingcomposition comprising a cross-linked polysaccharide backbone moleculehaving a plurality of binding molecules and a plurality of fluorescentlabels.

BACKGROUND OF THE INVENTION

In the development of immunoassay systems, many performance requirementsneed be met. Assays need be sensitive enough to detect analyte at verylow levels in the subpicogram nanogram range. Total assay time needs tobe 15 minutes or less in order to provide timely results for patientmanagement in point of care situations, or to meet throughputrequirements for batch analyzers. In some cases, analyte panels wheremultiple assays are simultaneously performed with the same sample areadvantageous in order to minimize the turnaround time for results andtest costs.

Many immunoassays employ fluorescent labels because such labels offermany practical advantages. Compared to enzymes, fluorescent labels aremuch more stable and do not require an additional substrate reagent. Formultianalyte panels, fluorescent labels enable the use of discretebinding zones within a common reaction chamber since each binding zonecan be sequentially subjected to fluorescence excitation and emissionmeasurements without interference from adjacent binding zones. Assaysutilizing fluorescent labels, however, are less sensitive than enzymebased assays primarily due to the enzyme's ability to catalyticallyconvert substrate to accumulate a great amount of product molecules overtime.

Arylsulfonate cyanine fluorescent dyes are described in Mujumdar et al.(1993) Bioconjugate Chemistry, 4:105-111; Southwick et al. (1990)Cytometry, 11:418-430; and U.S. Pat. No. 5,268,486. Cy5 is described ineach of the references and is commercially available from BiologicalDetection Systems, Inc., Pittsburgh, Pa., under the tradenameFLUOROLINK™ Cy5™. The arylsulfonate cyanine fluorescent dyes have highextinction coefficients (typically from 130,000 L/mole to 250,000L/mole), good quantum yields, fluorescent emission spectra in a range(500 nm to 750 nm) outside of the autofluorescence wavelengths of mostbiological materials and plastics, good solubilities, and lownon-specific binding characteristics.

Despite these excellent properties, arylsulfonate cyanine fluorescentdyes suffer from certain limitations. In particular, these dyes have arelatively narrow Stokes shift which results in significant overlapbetween the excitation and emission spectra of the dye. The overlap ofexcitation and emission spectra, in turn, can cause self-quenching ofthe fluorescence when the dye molecules are located close to each otherwhen excited. Such self-quenching limits the number of arylsulfonate dyemolecules which can be conjugated to a single antibody molecule for usein immunoassays. In the case of Cy5, an exemplary arylsulfonate cyaninefluorescent dye, the Stokes shift is 17 nm (which is the differencebetween an excitation wavelength of 650 nm and an emission wavelength of667 nm). Optimal fluorescent yield is obtained when from two to four Cy5molecules are conjugated to a single antibody molecule. The fluorescentsignal output drops rapidly when more than four dye molecules areconjugated to a single antibody molecule. The inability to conjugatemore than four dye molecules to individual antibody moleculessignificantly limits the sensitivity of immunoassays using Cy5-labelledantibodies and other binding substances.

There is a need for an improved optical detection system and an improvedmethod for detecting analytes with high sensitivity by fluorescentimmunoassay. The system and method should be easy to handle by the usersand provide high specific signal and minimal background noise.

SUMMARY OF INVENTION

The present invention is directed to a fluorescent detection system formeasuring a fluorescent signal on a probe tip. The system comprises: (a)a probe having an aspect ratio of length to width at least 5 to 1, theprobe having a distal end and a proximal end, the proximal end having asensing surface bound with a fluorescent label; (b) a light source foremitting excitation light directly to the probe's sensing surface; (c) acollecting lens pointed toward the sensing surface; and (d) an opticaldetector for detecting the emission fluorescent light; where thecollecting lens collects and directs the emission fluorescent light tothe optical detector.

The present invention is also directed to methods for detecting analytesby a fluorescent immunoassay. In one embodiment (three-step binding),the method comprises the steps of: (a) obtaining a probe having a firstantibody immobilized on the tip of the probe, wherein the diameter ofthe tip surface is ≤5 mm; (b) dipping the probe tip into a sample vesselcontaining a sample solution having an analyte, moving the probe tip upand down and flowing the sample solution laterally in the sample vessel;(c) dipping the probe tip into a reagent vessel containing a reagentsolution comprising a second antibody molecules conjugated with a firstmember of a binding pair, moving the probe tip up and down and flowingthe reagent solution laterally in the reagent vessel; (d) dipping theprobe tip into a washing vessel containing a wash solution; (e) dippingthe probe tip into an amplification vessel containing an amplificationsolution comprising a polymer having a molecular weight of at leastabout 1 million Dalton and conjugated with at least 5 molecules ofsecond member of the binding pair and at least 25 fluorescent labels,moving the probe tip up and down and flowing the amplification solutionlaterally in the amplification vessel to form an immunocomplex among theanalyte, the first antibody, the second antibody, and the first and thesecond members of the binding pair on the probe tip; (f) dipping theprobe tip into a second washing vessel containing a second washsolution; and (g) detecting the immunocomplex formed by detecting thefluorescent signal on the probe tip; wherein the first antibody and thesecond antibody are antibodies against the analyte.

The methods of the present invention achieves high sensitivity becausethe unique combination of (i) using a probe having a small sensingsurface area for binding analyte molecules, (ii) moving the probe tip upand down and flowing the reaction solution laterally in a reactionvessel, and (iii) using a high molecular weigh polymer conjugated withmultiple binding molecules and multiple fluorescent labels.

The present invention is further directed to a fluorescent labelingcomposition comprising: (a) a crosslinked FICOLL® (copolymers of sucroseand epichlorohydrin) having a molecular weight of at least 1 millionDaltons, (b) at least 5 binding molecules, and (c) at least 25fluorescent dye molecules, wherein the binding molecules and thefluorescent dye molecules are attached to the cross-linked FICOLL®.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a first embodiment of the optical detection system.

FIG. 2 illustrates a second embodiment of the optical detection system.

FIG. 3 illustrates a third embodiment of the optical detection system.

FIG. 4 illustrates a forth embodiment of the optical detection system.

FIG. 5 illustrates a fifth embodiment of the optical detection system.

FIG. 6 illustrates a sixth embodiment of the optical detection system.

FIG. 7 illustrates the two-step binding method for detecting an analyte.

FIG. 8 illustrates an optical detecting system where the light sourceand the detector are both mounted at the side opposite to the sensingsurface of the probe.

FIG. 9 illustrates another view of the optical detecting system wherethe probe is immersed into an analyte solution.

FIG. 10 illustrates a flow chart of preparing crosslinked FICOLL® 400.

FIG. 11 illustrates a flow chart of preparing Cy 5-antibody-crosslinkedFICOLL® 400.

FIG. 12 illustrates the elution pattern of SPDP-labeled crosslinkedFICOLL® 400 by Sepharose 4B CL chromatography.

FIG. 13 illustrates an immunoassay format for detecting protein A. Ab:antibody, Ag: antigen (protein A), Sa: streptavidin, B: biotin, F:fluorescent label.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used in the claims and specification are to be construed inaccordance with their usual meaning as understood by one skilled in theart except and as defined as set forth below.

“About,” as used herein, refers to within ±15% of the recited value.

An “analyte-binding” molecule, as used herein, refers to any moleculecapable of participating in a specific binding reaction with an analytemolecule. Examples include but are not limited to, (i) antigenmolecules, for use in detecting the presence of antibodies specificagainst that antigen; (ii) antibody molecules, for use in detecting thepresence of antigens; (iii) protein molecules, for use in detecting thepresence of a binding partner for that protein; (iv) ligands, for use indetecting the presence of a binding partner; or (v) single strandednucleic acid molecules, for detecting the presence of nucleic acidbinding molecules.

An “aspect ratio” of a shape refers to the ratio of its longer dimensionto its shorter dimension.

A “binding molecular,” refers to a molecule that is capable to bindanother molecule of interest.

“A binding pair,” as used herein, refers to two molecules that areattracted to each other and specifically bind to each other. Examples ofbinding pairs include, but not limited to, an antigen and an antibodyagainst the antigen, a ligand and its receptor, complementary strands ofnucleic acids, biotin and avidin, biotin and streptavidin, lectin andcarbohydrates. Preferred binding pairs are biotin and streptavidin,biotin and avidin, fluorescein and anti-fluorescein,digioxigenin/anti-digioxigenin. Biotin and avidin, including biotinderivatives and avidin derivatives such as streptavidin, may be used asintermediate binding substances in assay protocols employing complexbinding sequences. For example, antibodies may be labeled with biotin(“biotinylated”) and used to bind to a target substance previouslyimmobilized on a solid phase surface. Fluorescent compositions accordingto the present invention employing an avidin or streptavidin may then beused to introduce the fluorescent label.

“Immobilized,” as used herein, refers to reagents being fixed to a solidsurface. When a reagent is immobilized to a solid surface, it is eitherbe non-covalently bound or covalently bound to the surface.

“A monolithic substrate,” as used herein, refers to a single piece of asolid material such as glass, quartz, or plastic that has one refractiveindex.

A “numerical aperture,” as used herein, refers to a dimensionless numberthat characterizes the range of angles over which the system can acceptor emit light.

“An optical fiber,” as used herein, is a glass or plastic fiber thatcarries light along its length. An optic fiber is typically a circularcross-section dielectric waveguide consisting of a dielectric material(a core material) surrounded by another dielectric material with a lowerrefractive index (cladding).

A “probe,” as used herein, refers to a substrate coated with a thin-filmlayer of analyte-binding molecules at the sensing side. A probe has adistal end and a proximal end. The proximal end (also refers to probetip in the application) has a sensing surface coated with a thin layerof analyte-binding molecules.

Fluorescent Detection System

The present invention is directed to a fluorescent detection system formeasuring a fluorescent signal on a probe tip. The inventors havediscovered that by mounting both the light source and the detector atthe proximal side of the sensing surface of the probe, the undesiredreflection entering the detection optics is reduced and the detectionefficiency is increased.

The system comprises: (a) a probe having an aspect ratio of length towidth at least 5 to 1, the probe having a first end and a second end,the second end having a sensing surface bound with a fluorescent label;(b) a light source for emitting excitation light directly to the probe'ssensing surface; (c) a collecting lens pointed toward the sensingsurface; and (d) an optical detector for detecting the emissionfluorescent light; where the collecting lens collects and directs theemission fluorescent light to the optical detector

The probe can be a monolithic substrate or an optical fiber. The probecan be any shape such as rod, cylindrical, round, square, triangle,etc., with an aspect ratio of length to width of at least 5 to 1,preferably 10 to 1. Because the probe is dipped in a sample solution andone or more assay solutions during an immunoassay, it is desirable tohave a long probe with an aspect ratio of at least 5 to 1 to enable theprobe tip's immersion into the solutions. Heterogeneous assays can beperformed where the long probe is transferred to different reactionchambers. Dispensing and aspirating reagents and sample during the assayare avoided. The sensing surface of the probe is coated withanalyte-binding molecules and bound with fluorescent labels.

Any light source that can emit proper excitation light for thefluorescent label is suitable for the present invention. A prefer lightsource is a laser that can emit light with wavelengths suitable forfluorescent labels. For example, the laser center wavelength ispreferred to be 649 nm for Cy5 fluorescent dye. A suitable opticaldetector for detecting emission light is a photomultiplier tube (PMT), acharge coupled device (CCD), or a photodiode.

The light source and the optical detector including the collecting lensare mounted on the same side of the probe tip surface (the sensingsurface). If the sensing surface faces down, they are both mounted belowthe tip surface. If the sensing surface faces up, they are both mountedabove the tip surface. They are closer to the sensing surface than theother end of the probe. The sensing surface is always within the numericaperture of the collecting lens. The probe can be, but does not have tobe centrally aligned with the collecting lens.

FIG. 1 shows a first embodiment. An optically transparent rod (probe) ismade from glass or quartz. The lower end of the rod is used as a sensingsurface. Fluorescent labels are bond to the sensing surface. To detectthe fluorescence, the rod's sensing end is immersed into a vessel with aclear bottom that contains a buffer solution optimized for fluorescentemission. The clear bottom's material may be selected from plastic,glass or quartz. An optical detector and an excitation laser are mountedon the same side of the vessel, and the collecting lens is underneaththe sensing surface. The laser is aligned so that the laser beamprojected onto the rod sensing surface at an incident angle α, which isset greater than the rod's numerical aperture angle θ. Because the rod'srefractive index is much greater than the air and closer to that of thebuffer solution, a large portion of the incident laser light will passthrough the rod. A smaller amount of the laser light is reflected at thesensing surface. Some laser light is coupled into the rod and thenreflected back from the other end of the rod. When α>θ, the reflectedlight exits the rod's sensing surface to form a ring shaped light band.The center of this ring has much lower light intensity. As thecollecting lens is placed in the middle of the ring, the undesirablereflection entering the detection optics is reduced. To further decreasethis reflection, the upper end of the rod can be tapered and sanded tocertain roughness. To detect the emission at the sensing surface, aphoto multiplier tube or CCD can be used. The distance between the rod'ssensing surface and the collecting lens is adjustable to achieve thebest detection efficiency.

FIG. 2 shows a second embodiment where the rod is measured in airinstead of in a buffer solution contained in a vessel. The configurationof the laser and the detector is similar to the first embodiment, wherean optical detection system and an excitation laser are mounted on thesame side of the rod and the collecting lens is underneath the sensingsurface. The laser is aligned so that the laser beam projected onto therod sensing surface at an incident angle α, which is set greater thanthe rod's numerical aperture angle θ. Some incident laser light willpass through the rod. A great portion of the laser light is reflected atthe sensing surface. The remaining laser light is coupled into the rodand then reflected back from the upper end of the rod. When α>θ, thereflected light exits the rod's sensing surface to form a ring shapedlight band. The center of the ring has much lower intensity. As thecollecting lens is placed in the middle of the ring, the undesirablereflection entering the detection optics is avoided. To further decreasethis reflection, the upper end of the rod can be tapered and sanded tocertain roughness. To detect the emission at the sensing surface, aphoto multiplier tube or CCD can be used. The distance between the rod'ssensing surface and the collecting lens is adjustable to achieve thebest detection efficiency.

FIG. 3 shows a third embodiment that replaces the transparent rod of theembodiment in FIG. 1 with a non-transparent rod. The material of thenon-transparent rod is possibly plastic, ceramics, and metals. The lowerend of the rod is used as a sensing surface. To detect the fluorescence,the rod's sensing surface is immersed into a vessel with a clear bottomthat contains a buffer solution optimized for fluorescent performance.The clear bottom's material may be selected from plastic, glass orquartz. An optical detection system and an excitation laser are mountedon the same side of the rod the vessel, and the collecting lens isunderneath the sensing surface. The laser is aligned so that the laserbeam projected onto the rod's sensing surface at an incident angle α.Because the rod is non-transparent, the laser beam is reflected andabsorbed at the sensing surface. It is preferred that the rod's materialemits minimal fluorescence to the excitation laser. To detect theemission at the sensing surface, a photo multiplier tube or CCD can beused. The distance between the rod's sensing surface and the collectinglens is adjustable to achieve the best detection efficiency.

FIG. 4 shows a fourth embodiment that has the non-transparent rodmeasured in air instead of in a buffer solution contained in a vessel.The material of the non-transparent rod is possibly plastic, ceramic,and metal. The lower end of the rod is used as a sensing surface. Anoptical detection system and an excitation laser are mounted on the sameside the vessel, and the collecting lens is underneath the sensingsurface. The laser is aligned so that the laser beam projected onto therod's sensing surface at the incident angle α. Because the rod isnon-transparent, the laser beam is reflected and absorbed at the sensingsurface. It is preferred that the rod's material emits minimalfluorescence to the excitation laser. To detect the emission at thesensing surface, a photo multiplier tube or CCD can be used. Thedistance between the rod's sensing surface and the collecting lens isadjustable to achieve the best detection efficiency.

FIG. 5 shows a fifth embodiment that has a mirror like thin-film coatedon the sensing surface of the rod. The rod can be either transparent ornon-transparent. The mirror-like thin film coating is used to reflectlight, either excitation or emission. The mirror coating can usealuminum, gold or silver. A second thin-film of SiO₂ is optionallycoated on top of the first thin-film. The material of thenon-transparent rod can be plastic, ceramic, or metal. The material ofthe transparent rod is chosen among glass, quartz, or plastic.Fluorescent labels are bond to the sensing surface. To detectfluorescence, the rod's sensing end is immersed into a vessel with aclear bottom that contains a buffer solution optimized for fluorescentperformance. The clear bottom's material can be selected from plastic,glass or quartz. An optical detection system and an excitation laser aremounted on the same side the vessel, and the collecting lens isunderneath the sensing surface. The laser is aligned so that the laserbeam projected onto the rod's sensing surface at an incident angle α.Because the rod's sensing surface is coated with a first mirror thinfilm, the laser beam is reflected at a reflection angle α. To detect theemission at the sensing surface, a photo multiplier tube or CCD can beused. The distance between the rod's sensing surface and the collectinglens is adjustable to achieve the best detection efficiency.

FIG. 6 shows a sixth embodiment that uses a mirror like thin-filmcoating on the sensing surface of the rod, but the measurement is donein air. The rod can be either transparent or non-transparent. Themirror-like thin film coating is used to reflect light, eitherexcitation or emission. The mirror coating can use aluminum, gold orsilver. Another thin-film of SiO₂ is optionally coated on top of thefirst mirror thin film. The material of the non-transparent rod ispossibly plastic, ceramic, and metal. The material of the transparentrod is chosen from glass, quartz, or plastics. Fluorescent labels arebond to the sensing surface. To detect the fluorescence, the rod'ssensing end, an optical detection system and an excitation laser aremounted on the same side of the rod's sensing surface, and thecollecting lens is underneath the sensing surface. The laser is alignedso that the beam projected onto the rod's sensing surface at an incidentangle α. Because the rod's sensing surface is coated with a mirror thinfilm, the laser beam is reflected. To detect the emission at the sensingsurface, a photo multiplier tube or CCD can be used. The distancebetween the rod's sensing surface and the collecting lens is adjustableto achieve the best detection efficiency.

Although FIGS. 2, 4, and 6 are illustrated in a way that the laser andthe optical detector are mounted below the probe tip, it should beunderstood that the probe can be flipped upside down and the laser andthe optical detector can be mounted above the sensing surface to detectthe fluorescent signal.

Detecting an Analyte by a Fluorescent Immunoassay

The present invention is also directed to methods of detecting ananalyte in a liquid sample by a fluorescent immunoassay. The inventorshave discovered that the combination of (i) using a probe having a smallsensing surface area for binding analyte molecules, (ii) moving theprobe tip up and down and flowing the reaction solution laterally in areaction vessel, and (iii) using a high molecular weigh polymerconjugated with at least 5 binding molecules and at least 25 fluorescentlabels, improves the sensitivity of detection level to pg/mL.

FIG. 7 illustrates one embodiment of the methods. In this embodiment(two-step binding), the method comprises the steps of: (a) obtaining aprobe having a first antibody immobilized on the tip (sensing surface)of the probe, wherein the diameter of the tip surface is ≤5 mm; (b)dipping the probe tip into a sample vessel containing a liquid samplehaving an analyte; (c) moving the probe tip up and down and flowing thereagent solution laterally in the sample vessel to bind the analyte withthe first antibody; (d) dipping the probe tip into a reagent vesselcontaining a reagent solution comprising a polymer having a molecularweight of at least 1 million Daltons and conjugated with at least 5second antibody molecules and at least 25 fluorescent labels; (e) movingthe probe tip up and down and flowing the reagent solution laterally inthe reagent vessel to form an immunocomplex of the analyte, the firstantibody, and the second antibody on the probe tip; (f) dipping theprobe tip into a washing vessel containing a wash solution, anddetecting the immunocomplex formed by detecting the fluorescent signalon the probe tip; wherein the first antibody and the second antibody areantibodies against the analyte. In the above method, an optional washingstep can be added after the binding step (c). This extra washing stepmay not be required because the amount of the carried-over solution isminimal due to a small binding surface area.

In another embodiment (two-step binding), the method comprises the stepsof: (a) obtaining a probe having a first antibody immobilized on the tipof the probe, wherein the diameter of the tip surface is ≤5 mm; (b)dipping the probe tip into a sample vessel containing (i) a liquidsample having an analyte and (ii) a reagent solution comprising apolymer having a molecular weight of at least about 1 million Dalton andconjugated with at least 5 second antibody molecules and at least 25fluorescent label; (c) moving the probe tip up and down in the samplevessel and flowing the reagent solution laterally to form animmunocomplex of the analyte, the first antibody, and the secondantibody on the probe tip; (d) dipping the probe tip into a washingvessel containing a wash solution; and (e) detecting the immunocomplexformed by detecting the fluorescent signal on the probe tip; wherein thefirst antibody and the second antibody are antibodies against theanalyte.

In yet another embodiment (three-step binding), the method comprises thesteps of: (a) obtaining a probe having a first antibody immobilized onthe tip of the probe, wherein the diameter of the tip surface is ≤5 mm;(b) dipping the probe tip into a sample vessel containing a samplesolution having an analyte, moving the probe tip up and down and flowingthe sample solution laterally in the sample vessel; (c) dipping theprobe tip into a reagent vessel containing a reagent solution comprisinga second antibody molecules conjugated with a first member of a bindingpair, moving the probe tip up and down and flowing the reagent solutionlaterally in the reagent vessel; (d) dipping the probe tip into anamplification vessel containing an amplification solution comprising apolymer having a molecular weight of at least about 1 million Dalton andconjugated with at least 5 molecules of second member of the bindingpair and at least 25 fluorescent labels, moving the probe tip up anddown and flowing the amplification solution laterally in theamplification vessel to form an immunocomplex among the analyte, thefirst antibody, the second antibody, and the first and the secondmembers of the binding pair on the probe tip; (e) dipping the probe tipinto a second washing vessel containing a second wash solution; and (f)detecting the immunocomplex formed by detecting the fluorescent signalon the probe tip; wherein the first antibody and the second antibody areantibodies against the analyte. In the above method, optional washingsteps can be added after the binding steps (b) and (c). The extrawashing steps may not be required because the amount of the carried-oversolution is minimal due to a small binding surface area.

Methods to immobilize reagents to the solid phase (the sensing surfaceof the probe tip) are common in immunochemistry and involve formation ofcovalent, hydrophobic or electrostatic bonds between the solid phase andreagent. Analyte-binding molecules can be directly immobilized on thesensing surface. Alternatively, analyte-binding molecules can beindirectly immobilized on the sensing surface through a binding pair.For example, anti-fluorescein can be first immobilized either byadsorption to the solid surface or by covalently binding toaminopropylsilane coated on the solid surface. Then the analyte-bindingmolecule that is labeled with fluorescein can be bound to the solidsurface through the binding of fluorescein and anti-fluorescein (bindingpair).

The methods of the present invention achieves high sensitivity becausethe unique combination of (i) using a probe having a small sensingsurface area for binding analyte molecules, (ii) moving the probe tip upand down and flowing the reaction solution laterally in a reactionvessel, and (iii) using a high molecular weight polymer conjugated withmultiple binding molecules and multiple fluorescent labels.

The first factor of the present invention is to use a probe that has asmall tip for binding analytes. The tip has a smaller surface area witha diameter ≤5 mm, preferably ≤2 mm or ≤1 mm. The small surface of theprobe tip endows it with several advantages. In a solid phaseimmunoassays, having a small surface area is advantageous because it hasless non-specific binding and thus produces a lower background signal.Further, the reagent or sample carry over on the probe tip is extremelysmall due to the small surface area of the tip. This feature makes theprobe tip easy to wash, and causes negligible contamination in the washsolution since the wash solution has a larger volume. Another aspect ofthe small surface area of the probe tip is that it has small bindingcapacity. Consequently, when the probe tip is immersed in a reagentsolution, the binding of the reagent does not consume a significantamount of the reagent. The reagent concentration is effectivelyunchanged. Negligible contamination of the wash solution and smallconsumption of the reagents enable the reagent solution, theamplification solution, and the wash solution to be re-used many times,for example, 2-8 times.

However, binding reaction at the probe tip with a surface area is slow.When the probe tip is immersed in a solution, the ratio of bindingsurface area to solution volume is small, thus it demands a very longincubation time for target molecules to diffuse to the probe's sensingsurface. The second factor in the invention to enhance sensitivity is toinduce a lateral flow (orbital flow) of the solution across the probetip, which accelerates the capture of target molecules by its bindingpartner immobilized to solid phase. For example, the reaction vessel canbe mounted on an orbital shaker and the orbital shaker is rotated at aspeed at least 50 rpm, preferably at least 200 rpm, more preferably atleast 500 rpm, such as 500-1,000 rpm. Additionally, the probe tip ismoved up and down and perpendicular to the plane of the orbital flow, ata speed of 0.01 to 10 mm/second, in order to induce additional mixing ofthe solution above and below the probe tip. The combination of smallsurface for low background and flow for more rapid target moleculecapture produces high specific assay signals and low background noise,which are the determinants of analytical sensitivity.

The third factor in the invention to enhance sensitivity is the use ofhigh molecular weight polymers labeled with multiple binding moleculesand multiple fluorescent dyes. Fluorescent dyes have many practicaladvantages as labels in immunoassays; primarily they are very stable andeasy to link to binding proteins. Fluorescent dyes have a majorlimitation in that they cannot generate a sufficient fluorescent signalto be employed for sensitive assays. Therefore, it is important to havemultiple fluorescent dyes labeled on one polymer to increase thefluorescent signal. Many polymers such as dextran and FICOLL® andnucleic acid polymers are suitable as dye carriers. The fluorescentlabel can be attached directly to the polymer or it can be attachedindirectly to the polymer through a binding molecule such as an antibodyor streptavidin.

When the binding molecule is a polypeptide or protein, such as anantibody, the fluorescent label can covalently bind to it through avariety of moieties, including disulfide, hydroxyphenyl, amino,carboxyl, indole, or other functional groups, using conventionalconjugation chemistry as described in the scientific and patentliterature. Alternatively, antibodies can be biotinylated by knowntechniques (see Wilchek and Bayer, (1988) ANAL. BIOCHEM. 171:1-32) andlinked to the fluorescent label via avidin/streptavidin molecules.

Covalent binding of the fluorescent label to a polynucleotide can beeffected through a variety of moieties, including aldehyde, ketone,isothiocyanate, imidate, inosine, acyl, and alkyl, using conventionalconjugation chemistry, while derivatization with biotin is taught inmany references. (Leary et al. (1983) Proc. Natl. Acad. Sci. USA80:4045-4049; WO86/02929; EP063 879; Langer et al. (1981) Proc. Natl.Acad. Sci. USA 78:6633-6637; and EP2009 996).

Exemplary techniques for binding arylsulfonate cyanine fluorescent dyelabels to antibodies and other proteins are described in U.S. Pat. Nos.5,268,486; 5,650,334; the contents of which are in incorporated hereinby reference. Techniques for linking a preferred Cy5 fluorescent labelto both antibodies and nucleic acids are described in a technicalbulletin identified as Cat. No. A25000, published by BiologicalDetection Systems, Inc., Pittsburgh, Pa.

The methods of the present invention can be detected by the fluorescentdetection systems as described above in this application, where thelight source and the detector are mounted at the same side (the proximalside) of the sensing surface of the probe.

Alternatively, the methods of the present invention can be detected byan optical system where the light source and the detector are bothmounted at the side opposite to the sensing surface of the probe, andclose to the other end of the probe (FIG. 8-9)

In FIG. 8, the probe is removable from the detection system. The laserlight is directly coupled into the one end of the probe. The probe canmove in X-Y-Z directions in relative to the optical detection system.This motion allows the fine alignment between the probe and the opticaldetection system. The probe tip can be immersed into a well of astandard microtiter plate. The plate is mounted on an orbital shaker.The relative motion between the probe tip and the sample solution in thewell increases the assay speed.

As shown in an enlarged drawing in FIG. 9, the probe's tip is directlyimmersed into the analyte sample for binding assays. The coupling end isdetached from the optical detection system. A photo multiplier tube(PMT) is used as a photo detector.

The probe can be made of either a monolithic substrate or a fiber optic.If the probe is a fiber optic, then the fiber probe's coupling end canbe coated with an anti-reflection coating layer to improve the couplingefficiency.

In one embodiment, a polarized filter is placed in front of the laser,and a second polarized filter with perpendicular polarization is placedin front of the PMT. With two polarized filters, the undesirable laserreflection is minimized.

In another embodiment, the polarized filters can be replaced withbandpass filters that only allow the fluorescent wavelength to pass.

The probe's core is greater than 0.1 mm in diameter, but no greater than5 mm, preferably no greater than 2 mm, or 1 mm. A preferred numericalaperture is between 0.15 and 0.50.

High Molecular Weight Branched Polysaccharide Containing MultipleBinding Molecules and Fluorescent Labels.

The present invention is also directed to a fluorescent labelingcomposition comprising (a) a crosslinked FICOLL® having a molecularweight of at least 1 million Daltons, (b) at least 5 binding molecules,and (c) at least 25 fluorescent dye molecules, wherein the bindingmolecules and the fluorescent dye molecules are attached to thecross-linked FICOLL®. The composition preferably comprises 5-50 or 5-100binding molecules and 25-100 or 25-500 fluorescent dye molecules.

In one embodiment, the fluorescent dye molecules are attached directlyto the cross-linked FICOLL®. In another embodiment, the fluorescent dyemolecules are attached indirectly to the cross-linked FICOLL® throughthe binding molecules such as antibody molecules or streptavidins. Tominimize fluorescent quenching, the fluorescent dye molecules areattached to spaced-apart locations along the crosslinked FICOLL®.

This fluorescent labeling composition is a preferred composition for theabove-described methods of the present invention.

FICOLL® is commercially available in 70K and 400K Dalton molecularweights. FICOLL® offers advantages for serving as a macromolecularcarrier. One problem with crosslinking fluorescent protein in boostingassay sensitivity is that the non-specific, background signal can beincreased. Specific assay signal and background signal increasing in thesame proportion would result in the same signal to background ratio withno improvement in sensitivity. Polysaccharides in general exhibitnegligible non specific binding to many of the solid phase materialscommonly employed in immunoassays. Consequently, a FICOLL®macromolecular carrier increases the signal in specific binding whileminimizing the non specific binding to the solid phase, thereforeyielding an improvement in the signal to background ratio enhancingsensitivity.

FICOLL® is advantageous over an alternative polysaccharide dextran.Dextran, since it is linear with few branch points, is extremelypolydisperse in its molecular weight distribution. In linking protein topolysaccharide carriers, reproducible results are obtained when startingmaterial are of a defined, narrow molecular weight range. The branchedstructure of FICOLL® endows it with some tolerance to chemical cleavageor mechanical shearing and thus minimizing the impact on its molecularweight. The other aspect of the branched structure if FICOLL® is that itminimizes the interaction of the polymer with solid phase material usedin immunoassay; consequently non-specific binding is lower compared toother polysaccharides.

FICOLL® 400 (molecular weight) is polydisperse and the vast majority ofthe material actually fractionates as much smaller molecules than IgG.The ideal polysaccharide carrier should have a molecular weight greaterthan one million Daltons, which elutes at or near the void volume of aSepharose 4B CL column. Very little of the FICOLL® 400 preparationexhibits such high molecule weight polymers to be practically useful.

An aspect of the invention is to prepare crosslinked derivatives ofFICOLL® to create high molecular weight polymers. The crosslinking ofFICOLL® further increases the degree of branching within the polymer. Anadvantage of this aspect of the invention is that FICOLL® polymers canbe prepared in a wide range of molecular weights by controlling thecrosslinking chemistry. Crosslinked FICOLL® with molecular weightsgreater than one million Daltons can be achieved and those polymersremain soluble. Such polymers are capable of further derivatization forfluorescent dye and protein conjugation; the conjugated polymers exhibitsignal amplification in solid phase binding assays, and mostsurprisingly, low non-specific binding to the solid phase. Theseperformance features are not expected with commercial preparations ofFICOLL® 400 or other polysaccharides.

The crosslinked FICOLL® compositions should have the following features:(a) soluble at greater than 2.5, 5 or 10 mg/ml; (b) mean hydrodynamicdiameter greater than 100, 150, or 200 nm; (c) molecular weight greaterthan, 1, 2, 5, or 10 MD and (d) at least 25 functional groups perFICOLL® for attaching binding molecules and/or fluorescent dyemolecules.

Amine derivatives of FICOLL® are commercially available which enablescrosslinking. FIG. 10 illustrates a flow chart for preparing crosslinkedFICOLL®.

Many fluorophores are commercially available as NHS derivatives thatallow for coupling to the amine groups of proteins such as streptavidinor antibodies. Cy5 and Alexa Fluor 647 are good examples. Thefluorescent labeled protein is then coupled to an amine derivative ofFICOLL® by using the well established maleimide/thiol proteinconjugation procedure or other crosslinking methods. FIG. 11 illustratesa flow chart for antibody conjugation to crosslinked FICOLL®.

Crosslinked FICOLL® can serve as a macromolecule carrier of Cy 5anti-FITC and such a conjugate can be employed of boost immunoassaysensitivity compared to monomeric Cy 5-anti FITC.

The invention is illustrated further by the following examples that arenot to be construed as limiting the invention in scope to the specificprocedures described in them.

EXAMPLES Example 1. Preparation of Crosslinked FICOLL® 400

FIG. 10 shows a flow chart of preparing crosslinked Ficoll 400.

To 2 ml of FICOLL® 400 (Sigma/Aldrich) that was aminated to contain 88amines per FICOLL® 400 kD (Skold Technology) at 20 mg/ml in PBS wasadded 10 μL of SPDP (succinimydyl6-[3-[2-pyridyldithio]-proprionamido]hexanoate, Invitrogen) at 50 mg/mlin DMF (N,N-Dimethylformamide). The SPDP to FICOLL® molecular couplingratio (MCR) was 15. The mixture reacted for 1 hour at room temperatureand followed by dialysis. Thiol incorporation was estimated to be 5.5per FICOLL® 400 kD by standard methods.

To deprotect the thiols on SPDP-labeled FICOLL® 400, 30 μL of DTT(dithiotheritol, Thermo Scientific) at 38 mg/ml PBS was added to 20 mgin 1 ml PBS and allowed to react for two hours at room temperature. TheSH-FICOLL® was purified on a PD10 column.

SMCC (succinimidyl 4[N-malemidomethyl]cyclohexan-1-carboxylate) waslinked to aminated FICOLL® 400 (88 amines/-FICOLL®) in two preparationsas follows: 1) Aminated Ficoll 400 at 10 mg in 1 ml PBS was mixed with25 μL SMCC (Pierce Chemical) at 10 mg/ml DMF for a SMCC/FICOLL® MCR of30. The mixture reacted for two hours at room temperature and followedby purification on a PD10 column (GE Healthcare). 2) Aminated FICOLL®400 at 10 mg in 1 ml PBS was mixed with 12.5 μL SMCC at 10 mg/ml DMF fora SMCC/FICOLL® MCR of 15. The mixtures reacted for 2 hours at roomtemperature followed by purification on a PD10 column.

To crosslink the SH-FICOLL® 400 and SMCC-FICOLL® 400 two preparationswere made: 1.) 10 mg in 1 ml PBS SH-FICOLL® 400 was mixed with 10 mg in1 ml PBS SMCC-FICOLL® 400 (30 MCR). 2.) 10 mg in 1 ml PBS SH-FICOLL® 400was mixed with 10 mg in 1 ml PBS SMCC-FICOLL® 400 (15 MCR). The mixturesreacted for overnight at 30° C.

To provide linking sites for antibody conjugation to the crosslinkedFICOLL® 400, the residual amines were then reacted with an excess ofSPDP. 20 mg of crosslinked FICOLL® 400 was mixed with 75 μL SPDP at 50mg/ml DMF. The mixture reacted for 1 hour at room temperature followedby dialysis versus PBS.

The SPDP labeled crosslinked FICOLL® 400 preparations were thenfractionated on a Sepharose 4B CL (GE Healthcare) column. The resultsshow that the crosslinked FICOLL® are much larger polymers than thenative FICOLL® 400, and the extent of crosslinking is dependent on theMCR of SMCC. High molecular weight polymers, which were eluted atfractions 40-50 with the peak at about fraction 45 eluting at or nearthe void volume of the 4B CL, were achieved with a SMCC MCR of 30 (FIG.12); they are the preferred polymers for subsequent conjugation tobinding proteins and fluorescent dye carrier. For comparison, the voidfraction was fraction 32, and the non-crosslinked SPDP-FICOLL® washighly dispersed and eluted at fractions 50-120 with the peak at aboutfraction 98.

Example 2. Preparation of Cy5-Antibody-Crosslinked FICOLL® Conjugates

FIG. 11 shows a flow chart of preparing Cy 5-antibody-crosslinkedFICOLL® 400. Anti FITC (Biospacific) at 3.2 mg/ml in 1 ml 0.1 M sodiumcarbonate pH 9.5 was mixed with 10.6 μl Cy5-NHS (N-Hydroxysuccinimide,GE Healthcare) at 10 mg/ml DMF and allowed to react for ½ hour at 30° C.The mixture was then purified on a PD 10 column. The Cy5-anti FITC at1.5 mg/ml in 1 ml PBS was mixed with 1.9 μL SMCC at 5 mg/ml DMF andreacted for 1 hour at room temperature followed by purification on a PD10 column.

The thiols on crosslinked FICOLL® 400-SPDP were deprotected by adding 30μL DTT at 38 mg/ml to 0.7 mg crosslinked FICOLL® 400-SPDP in 1 ml PBSand reacting for 1 hour at room temperature followed by a PD 10 columnto purify the crosslinked FICOLL®.

The Cy5-anti FITC-SMCC was mixed with crosslinked FICOLL® 400-SH andreacted overnight at room temperature. 10 μL NEM (N-ethyl-maleimide,Aldrich) at 12.5 mg/ml was then added and reacted for ½ hour at roomtemperature. The conjugate was then purified on a Sepharose 4B CLcolumn.

Example 3. Preparation of Cy5-Antibody Dextran Conjugates

Cy5-antibody dextran (linear) conjugate (described in U.S. Pat. No.5,650,334) was prepared for comparative studies.

Anti-FITC (Biospacific) at 3.2 mg/ml in 1 ml 0.1 M sodium carbonate pH9.5 was mixed with 10.6 μL Cy5-NHS (GE Healthcare) at 10 mg/ml DMF andallowed to reacted for ½ hour at 30° C. The mixture was then purified ona PD 10 column.

The Cy5-anti FITC at 1.5 mg/ml in 1 ml PBS was mixed with 1.9 μL SMCC at5 mg/ml DMF and reacted for 1 hour at room temperature followed bypurification on a PD 10 column.

To thiolate dextran, 150 μL of a 34 mg/ml solution of succinimydyl6-[3-[2-pyridyldithio]-proprionamido]hexanoate (LC-SPDP) (Pierce #21651)in DMF was added to a 5 ml solution containing 20 mg/ml aminodextran(Molecular Probes, #D-7145, 130 amines/dextran, 2000 kD M.W.) in PBS atpH 7.4. The reaction proceeded for 30 minutes at room temperature (RT),then the mixture was dialyzed overnight at RT against PBS. Greater than95% of the amines in aminodextran were labeled with LC-SPDP during thereaction. The LC-SPDP-dextran was then purified on a Sepharose 4B CLcolumn. To remove the low molecular weight dextran fragments, only thefractions of the void peak were collected.

A 6.15 ml solution of Cy 5-anti FITC at 2.6 mg/ml in PBS pH 7.4 wasmixed with 156 of 2.7 mg/ml SMCC (Pierce #22320) in DMF and was allowedto react for 30 minutes at RT. Unreacted SMCC was removed bypurification on a PD 10 column.

The LC-SPDP-dextran was reduced by adding 156 μL of 0.5M dithiothreitolto 5.2 ml of 3.1 mg/ml LC-SPDP-dextran and incubating for 15 minutes atRT. The dextran was then purified on a PD-10 (Pharmacia #17-0851-01)column.

Conjugation was achieved by mixing the reduced LC-SPDP-dextran with theSMCC-anti-FITC and incubating overnight at RT. NEM (Sigma #12828-7) wasadded at a final concentration of 1.0 mM to stop the reaction. Theconjugate was then applied to a Sepharose 4B CL column and fractionseluting in the void volume were collected.

Example 4. Troponin I Assay Materials

Reaction disks made from PMMA plastic having benzophenone bovine serumalbumin (BSA)-biotin immobilized on the bottom were prepared as follows.100 μL benzophenone BSA-biotin per disk was then immobilized at 10 μL/mlby UV-curing for 60 minutes.

Streptavidin-monoclonal anti TnI conjugate (SA-anti TnI) was preparedusing heterobifunctional linking reagents, S-acetylthioglycolic acidN-hydroxysuccinimide ester (SATA, Pierce #26102), and SMCC (Pierce#22320). Fifteen (15) molar excess of SATA (dissolved at 5 mg/mL in DMF)was reacted with 1 mg of anti-TnI (BioDesign) at 0.74 mg/mL in PBS at pH7.4 for 3 hours at room temperature. Also, 15 molar excess of SMCC(dissolved at 5 mg/mL in DMF) was reacted with 1.1 mg of streptavidin at1.1 mg/mL in PBS for 3 hours at room temperature. Unreacted linkers wereremoved from both anti TnI/SATA and streptavidin/SMCC reaction mixturesusing a PD 10 column. The purified antiTnI-SATA and streptavidin-SMCCwere mixed at the protein molar ratio of 1:3. The conjugation reactionwas initiated by adding 1M hydroxylamine to a final concentration of 100mM and incubated over 18 hours at 4° C. The reaction was stopped byadding 100 mM NEM (Aldrich Chemical) at a final concentration of 1 mM inthe reaction mixture and incubating for 15 minutes at room temperature.After the incubation with NEM, mixture was purified using Sephacryl S300 column (GE Healthcare).

40 μL of SA-anti-TnI at 25 μg/ml was added to each disks and incubatedfor 1 hour at room temperature. The assay buffer was PBS, 1% BSA, 0.1%Tween 20, pH 7.4. The disks were then washed 3 times.

Affinity purified goat anti-TnI peptide 3 (Biospacific) was labeled withfluorescein as follows: 1 mg of antibody in 1.8 ml PBS was mixed with 64μL fluorescein-NHS (Invitrogen) at 2 mg/ml DMF and reacted to 2 hours atroom temperature followed by purification on a PD 10 column.

Example 5. Comparison of Cy 5-Anti FITC Vs. Cy 5-Anti FITC-CrosslinkedFICOLL® in Troponin I Immunoassay

Troponin I at 0, 10 and 50 ng/ml in 40 μL sample volumes were added todisks and incubated for 1 hour at room temperature. The disks werewashed three times in assay buffer. 40 μL of fluorescein labeledanti-TnI peptide 3 at 10 μg/ml was added to each disk and incubated for25 minutes at room temperature followed by three washes. 40 μL of eitherCy5-anti FITC, Cy5-anti FITC-crosslinked (cx) FICOLL®, each at 10 μL/mlof antibody, was added to the disks with a 25 minute incubation at roomtemperature, followed the three washes. Cy5 fluorescence was thenmeasured on the surface of each disk (Table 1).

TABLE 1 Assay Results Voltage Background Voltage Corrected Signal/NoisePlastic 0.9 Background Cy5-Anti FITC Tnl, 0 ng/ml 1.01 0.95 av. 0.980.09 1 Tnl, 10 ng/ml 1.28 1.18 av. 1.23 0.33 4.1 Tnl, 50 ng/ml 2.68 3.55av. 3.11 2.21 27.6 Cy5-Anti FITC-Cx-FICOLL ® Tnl, 0 ng/ml 1.02 0.97 av.0.99 0.09 1 Tnl, 10 ng/ml 3.15 3.15 av. 3.15 2.21 24.6 Tnl, 50 ng/ml8.95 8.93 av. 8.94 8.04 89.3

Example 6. Comparison of Cy 5-Anti FITC-Crosslinked FICOLL® Vs. Cy5-Anti FITC-Dextran in TnI Immunoassay

Troponin I at 0 100 ng/ml in 40 μl sample volumes were added to disksand incubated for 1 hour at room temperature. The disks were washedthree times in assay buffer. 40 μl of fluorescein labeled anti TnIpeptide 3 at 10 μg/ml was added to each disk and incubated for 120minutes at room temperature followed by three washes. 40 μL of eitherCy5-anti FITC-Dextran (Example 3) or Cy5-anti FITC-crosslinked FICOLL®(Example 2), each at 10 ug/ml of antibody, was added to the disks with a25 minute incubation at room temperature, followed the three washes. Cy5fluorescence was then measured on the surface of each disk (see Table2).

TABLE 2 Assay Results Voltage Background Voltage Corrected Signal/NoicePlastic 0.85 Background Cy5-Anti FITC-Dextran Tnl, 0 ng/ml 4.71 3.82 av.4.25 3.4 1 Tnl, 100 ng/ml 7.32 7.61 av. 7.45 6.61 1.9 Cy5-AntiFITC-Cx-FICOLL ® Tnl, 0 ng/ml 2.21 2.3 av. 2.25 1.4 1 Tnl, 100 ng/ml 8.56.9 av. 7.7 6.85 4.9

Example 7. Preparation of Cy5-Streptavidin-Crosslinked FICOLL®

Cy 5 Labeling of Streptavidin

32 μL of Cy 5-NHS (GE Healthcare) at 5 mg/ml in DMF reacted with 1 ml ofstreptavidin (Scripps Labs) at 2.4 mg/ml in 0.1 M sodium carbonatebuffer pH 9.5 for 40 minutes at 30° C. Applying the mixture to a PD 10column (Pharmacia) removed unconjugated Cy 5. Spectral analysisindicated 2.8 Cy 5 linked per streptavidin molecule.

Conjugation of Cy 5-Streptavidin to Crosslinked FICOLL®

5.8 μL of SMCC (Pierce Chemical) at 10 mg/ml in DMF reacted with 2 mgstreptavidin in 1 ml PBS pH 7.4 for 1 hour at room temperature. Applyingthe mixture to a PD 10 column removed unbound SMCC.

The thiols on crosslinked FICOLL® 400-SPDP were deprotected by adding 30μL DTT at 38 mg/ml to 1 mg crosslinked FICOLL® 400-SPDP in 1 ml PBS andreacting for 1 hour at room temperature followed by a PD 10 column topurify the crosslinked FICOLL®.

The Cy5-streptavidin-SMCC was mixed with crosslinked FICOLL® 400-SH andreacted overnight at room temperature. 10 μL NEM (Aldrich) at 12.5 mg/mlwas then added and reacted for ½ hour at room temperature. The conjugatewas then purified on a Sepharose 4B CL column.

Example 8. Preparation of Cy 5 Labeled Crosslinked FICOLL® andConjugation to Streptavidin

The above methods (Examples 2, 3 and 7) require the binding protein toundergo two chemical modifications, one Cy 5 labeling and a second withSMCC to enable conjugation to crosslinked FICOLL®. In some casesparticularly with antibodies, two chemical modifications may not bedesirable since a loss in binding activity could result. The followingmethod entails labeling crosslinked FICOLL® directly with Cy 5 followedby conjugation to the binding protein. The method therefore requiresonly a single chemical modification of the binding protein.

Preparation of Cy 5 Labeled Crosslinked-FICOLL®

To 2 ml of FICOLL® 400 (Sigma/Aldrich) that was aminated to contain 88amines per FICOLL® 400 kD (Skold Technology) at 20 mg/ml in PBS wasadded 10 μL of SPDP (Invitrogen) at 50 mg/ml in DMF. The SPDP to FICOLL®molecular coupling ratio (MCR) was 15. The mixture reacted for 1 hour atroom temperature followed by dialysis. Thiol incorporation was estimatedto be 5.5 per FICOLL® 400 kD by standard methods.

To deprotect the thiols on SPDP labeled FICOLL® 400, 30 μL of DTT(Thermo Scientific) at 38 mg/ml PBS was added to 20 mg in 1 ml PBS andallowed to react for two hours at room temperature. The SH-FICOLL® waspurified on a PD 10 column.

SMCC was linked to aminated FICOLL® 400 (88 amines/Ficoll) as follows:Aminated FICOLL® 400 (88 amines/FICOLL®) at 10 mg in 1 ml PBS was mixedwith 25 μl SMCC (Pierce Chemical) at 10 mg/ml DMF for a SMCC/FICOLL® MCRof 30. The mixture reacted for two hours at room temperature followed bypurification on a PD10 column

To crosslink the SH-FICOLL® 400 and SMCC-FICOLL® 400, 10 mg ofSH-FICOLL® 400 in 1 ml PBS was mixed with 10 mg in 1 ml PBS ofSMCC-FICOLL® 400 (30 MCR). The mixture reacted for overnight at 30 C.

To provide linking sites for protein conjugation to the crosslinkedFICOLL® 400, the residual amines were then reacted with SPDP at a MCR of30. 20 mg of crosslinked FICOLL® 400 was mixed with 64 μl SPDP at 10mg/ml DMF. The mixture reacted for 1 hour at room temperature followedby dialysis versus PBS. Modification with SPDP at a MCR of 30 leavessufficient number to amino groups for subsequent labeling with Cy5-NHS.The SPDP labeled crosslinked FICOLL® 400 preparation was thenfractionated on a Sepharose 4B CL (GE Healthcare) column.

For Cy 5 labeling, 15 μL of Cy 5-NETS at 5 mg/ml in DMF reacted with 1mg SPDP-crosslinked FICOLL® in 1 ml 0.1 M sodium carbonate pH 9.0 for 1hour at room temperature. The mixture was purified in a PD 10 column.Assuming the crosslinked FICOLL® had an average molecular weight of 4million Daltons, spectral analysis showed about 45 Cy 5 incorporated percrosslinked FICOLL®. Antibody and streptavidin are typically labeledwith about 2 Cy5.

Conjugation of Streptavidin to Cy 5-Crosslinked FICOLL®

5.8 μL of SMCC (Pierce Chemical) at 10 mg/ml in DMF reacted with 2 mgstreptavidin in 1 ml PBS pH 7.4 for 1 hour at room temperature. Applyingthe mixture to a PD 10 column removed unbound SMCC.

The thiols on Cy 5-crosslinked FICOLL® 400-SPDP were deprotected byadding 30 DTT at 38 mg/ml to 0.7 mg crosslinked FICOLL® 400-SPDP in 1 mlPBS and reacting for 1 hour at room temperature followed by a PD 10column to purify the crosslinked FICOLL®.

The streptavidin-SMCC was mixed with Cy 5-crosslinked FICOLL® 400-SH andreacted overnight at room temperature. 10 μL NEM (Aldrich) at 12.5 mg/mlwas then added and reacted for ½ hour at room temperature. The conjugatewas then purified on a Sepharose 4B CL column.

Example 9. Protein A Immunoassay Probe Format

Probe Preparation

Quartz probes, 1 mm diameter and 2 cm in length, were coated withaminopropylsilane using a chemical vapor deposition process (YieldEngineering Systems, 1224P) following manufacturer's protocol. The probetip was then immersed in a solution of murine monoclonalanti-fluorescein (Biospacific), 10 μg/ml in PBS at pH 7.4. Afterallowing the antibody to adsorb to the probe for 5 minutes, the probetip was washed in PBS. The probe tip was then immersed in a solutioncontaining affinity purified fluorescein labeled chicken anti-Protein A(Cygnus Technologies) at 10 μg/ml in PBS. The antibody wasfluoresceinated by a standard method. After 10 minutes the probe tip waswashed in PBS.

Protein A Immunoassay

The assay format is illustrated in FIG. 13.

The anti-Protein A coated probe tip was immersed in a microwellcontaining 200 μL Protein A samples diluted in assay buffer (PBS, 10mg/ml BSA, 0.1% Tween 20). The microwells were positioned on an orbitalmixer (Big Bear Automation) and with the probe held stationary, themicrowell were moved at 250 rpm with a 2 mm diameter orbital stroke.After 30 minutes the probe tips were washed 3 times with PBS. The probetips were then immersed in a microwell with 200 μL of biotin labelaffinity purified chicken anti-Protein A (Cygnus Technologies) at 10μg/ml in assay buffer. The anti-Protein A was biotinylated by a standardmethod. After 5 minute incubation with orbital flow at 250 rpm, theprobes were washed 3 times with PBS. The probe tips were them immersedin a microwell containing Cy5-streptavidin-crosslinked FICOLL® at 10μg/ml in assay buffer. After five minute incubation at 250 rpm orbitalflow, the probe tips were washed 3 times with PBS. Cy5 fluorescencebound to the probe tip was measured with the optics depicted in FIG. 1.Table 3 shows the results.

TABLE 3 Assay Results Pro A mV Av.   0 pg/ml 93 92 87 91 78 78 76 73 77108 96 94 112 109 85 102 62 65 57 61 98 108 104 103 Av. 87  10 pg/ml 119150 131 133 128 139 127 131 155 147 145 149 152 140 118 137 Av. 138  100pg/ml 349 376 364 360 317 320 293 310 Av. 335 1000 pg/ml 1184 1167 11521168 1300 1447 1456 1401 1163 1146 1123 1144 1168 1004 1011 1061 18291312 1536 1559 1600 1124 1061 1262 Av. 1266

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thisspecification.

What is claimed is:
 1. A detection system for measuring a fluorescentsignal in a fluorescent assay, comprising: a probe having an aspectratio of length to width at least 5 to 1, the probe having a distal endand a proximal end, the proximal end having a sensing surface bound witha fluorescent label; a light source for emitting excitation lightdirectly to the probe's sensing surface; a collecting lens pointedtoward the sensing surface; and an optical detector for detecting theemission fluorescent light; where the collecting lens collects anddirects the emission fluorescent light to the optical detector.
 2. Thesystem according to claim 1, wherein the probe sensing surface has adiameter equal or less than 5 mm.
 3. The system according to claim 1,wherein the probe is transparent.
 4. The system according to claim 3,wherein the distal end of the probe is tapered and/or roughened toreduce undesired reflection.
 5. The system according to claim 3, whereinthe light source is aligned so that the light beam projected onto thesensing surface at an incident angle greater than the numerical apertureangle of the probe.
 6. The system according to claim 3, wherein theprobe is made of glass, quartz, or plastic.
 7. The system according toclaim 1, wherein the probe is a non-transparent monolithic rod.
 8. Thesystem according to claim 1, wherein the probe is made of plastic,ceramic, or metal.
 9. The system according to claim 1, wherein thesensing surface is coated with a film selected from aluminum, gold, orsilver, the film has a thickness from about 50 nm to about 500 μm. 10.The system according to claim 9, wherein a second film of SiO₂ is coatedon top of the film of claim
 9. 11. The system according to claim 1,further comprising a vessel containing a solution, wherein the proximalend of the probe is immersed in the solution, and the light source andthe collecting lens are mounted on the bottom side of the vessel.